1. Field of the Invention
The invention relates to a process for the purification and recovery of formate-dehydrogenase from Candida boidinii
2. Brief Description of the Prior Art
Formate-dehydrogenase (FDH) is present intracellularly in Candida boidinii. Hitherto two different processes have been used to recover FDH. Thus, in Eur. J. Biochem., 62 (1976), 151-160 a process is described in which Candida boidinii is cultured, the harvested cells are frozen, left to stand in an aqueous phosphate-containing medium and then mechanically disintegrated, the cell debris is centrifuged off, residual protein is precipitated with Streptomycin sulphate and the precipitate is centrifuged off, after which the supernatant solution is subjected to ion-exchange chromatography on DEAE cellulose to recover the enzyme. There is also known, from J. Chem. Tech. Biotechnol., 32 (1982) 130 to 137, a process in which Candida boidinii is cultured, the harvested cells are mechanically disintegrated, the resulting cell suspension is subjected to heat-denaturation and the resulting suspension is subjected to four successive phase distribution steps, using aqueous 2-phase systems with polyethylene glycol and potassium phosphate as phase formers.
In addition, a series of papers on the affinity distribution of non-intracellular enzymes in phase systems containing triazine dyestuffs that are bonded to polyethylene glycol have been disclosed; cf., for example, Kroner et al. in Gribnau et al., Affinity chromatography and related techniques, [pages 491 to 501], Elsevier, Amsterdam 1982; Cordes et al. in Publ. 3rd Eur. Congr. Biotechnology, Munich 1984, Vol III, pages 557 to 564; Eur. J. Biochem., 131 (1983) 589 to 594; J. Chromatography, 259 (1983) 97 to 105; Analytical Biochemistry, 136 (1984) 264 to 271; and J. Chromatography, 298 (1984) 483 to 493. Finally, in Analytical Biochemistry, 124 (1982) 117 to 124 a process is described in which baker's yeast is used as the starting material, and phosphofructokinase, used as the non-intracellular enzyme, is precipitated with polyethylene glycol and subsequently subjected to a phase distribution in an aqueous 2-phase system, the system comprising dextran, polyethylene glycol and Cibacron-blue-S3G-A as the triazine dyestuff that is bonded to polyethylene glycol.
With the known processes for the purification and, if desired, recovery of FDH there is a need, however, to reduce the number of purification steps.